Abstract

Current antibody-drug conjugates (ADC) have made advances in engineering the antibody, linker, conjugation site, small molecule payload and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADC is poorly understood. Here we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, co-administration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared to T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amount of payload delivered to each targeted cell but increasing the number of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist in vitro and has no single-agent efficacy in vivo, yet improves the effectiveness of T-DM1 in vivo. Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metabolism and confirmed that the in vivo cellular dose of T-DM1 alone exceeded the minimum required for efficacy in this model. Additionally, this technique characterized cellular pharmacokinetics with heterogeneous delivery after one day, degradation and payload release by two days, and in vitro cell killing and in vivo tumor shrinkage 2-3 days later. This work demonstrates that the intratumoral distribution of ADC - independent of payload dose or plasma clearance - plays a major role in ADC efficacy.